Electrodes coated with treating agent and uses thereof

ABSTRACT

An object of the invention is to provide a method for delivery of macromolecules into biological cells in the tissues of a patient and includes the steps of: (a) providing electrodes ( 16 ) in an electrode assembly ( 12 ), wherein the electrodes have fixed electrode surfaces ( 42 ) which are coated with at least one static layer of electrode releasable molecules ( 44 ) to be delivered; (b) providing a waveform generator ( 15 ) for generating electric fields; (c) establishing electrically conductive pathways between the electrodes ( 16 ) and the waveform generator ( 15 ); (d) locating the electrodes ( 16 ) such that the biological cells are situated therebetween, and (g) providing electric fields in the form of pulse waveforms from the waveform generator ( 15 ) to the electrodes ( 16 ), such that molecules in the at least one static layer of the electrode releasable molecules ( 44 ) on the electrodes ( 16 ) are delivered into the biological cells. The electrode releasable molecules ( 44 ) can be either electric field separable molecules and/or solvent separable material. Another object of the invention is to provide an apparatus for carrying out the method of the invention. The static-coated electrode assembly ( 12 ) can be provided in a sterile package ( 24 ), from which the electrode assembly ( 12 ) is removed prior to use. The statically-coated electrode assembly ( 12 ) can be in a form of a disposable assembly ( 12 ) which is removable and replaceable from an electrode assembly holder ( 13 ).

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority based upon copending PCTInternational Application Number PCT/US00/00014, filed 12 Jan. 2000,which is based upon copending U.S. Provisional Application Serial No.60/117,755, filed 28 Jan. 1999, and which was published on 3 Aug. 2000with PCT International Publication Number WO 00/44438.

TECHNICAL FIELD

[0002] The present invention relates generally to methods and apparatusfor delivery of macromolecules into cells. More specifically, thepresent invention provides methods and apparatus for deliveringsubstances, such as macromolecules, e. g. deep tumor tissue treatingagents, polynucleotide vaccines (DNA vaccine and/or RNA vaccine) andprotein-based vaccines, into cells in tissues.

BACKGROUND ART

[0003] The first DNA vaccination procedure in the prior art was callednaked DNA vaccination because a liquid solution of DNA was injected intothe muscle of mice with no additives to enhance transfection. Thismethod does transfect a few cells and does induce an immune response tothe expressed antigen in mice. However, in humans and primates, themethod does not work well.

[0004] In the prior art, an improvement in DNA vaccine efficiency wasobtained by the use of a biolistic method for DNA delivery. Thebiolistic method is done by coating metal microbeads with DNA andshooting the particles into skin after accelerating the particles to achosen velocity. This method works much better than naked DNA. Part ofthe reason is that the DNA coated particles are injected into the skinto a depth that increases the chance of transfecting Langerhans cells.However, the biolistic method has some disadvantages. First, it causessome skin damage that may scar in some individuals. Second, in spite ofthe increased efficiency, more efficiency is needed. Third, theballistic particle remains inside the patient after treatment. In thisrespect, it would be desirable if a method for delivering DNA tobiological cells were provided which does not cause skin damage thatresults in scarring. Also, it would be desirable if a method fordelivering DNA to biological cells were provided which does not leave aresidue of ballistic particles in cells that are treated. As a matter ofinterest, the following U.S. patents disclose biolistic methods: U.S.Pat. Nos. 5,036,006 and 5,478,744.

[0005] A number of additional approaches to delivering macromolecules tobiological cells are disclosed in the prior art and are represented bythe following U.S. patents or other publications as follows.

[0006] U.S. Pat. No. 5,019,034 of Weaver et al discloses a process forelectroporation of tissues in which electrodes are placed on top of thetissue surface, such as skin, of a patient. Molecules that are used fortreating the skin are placed in reservoirs on top of the skin surface,and the treatment molecules must penetrate into the skin tissuestransdermally. That is, the treatment molecules must pass from outsidethe skin to inside the skin. Not only is the surface layer of the skinrelatively impermeable, if the layers of the skin to be treated are nearthe basal lamina of the epidermis, then the treatment molecules musttraverse considerable skin tissue before the cells to be treated arereached by the treatment molecules Such a treatment method isinefficient for treating cells near the basal lamina. Rather than usingelectrodes that are placed on the skin surface and have treatmentmolecules pass through the skin transdermally to treat biological cellsnear the basal lamina of the epidermis, it would be desirable if anelectroporation method were provided for delivering molecules tobiological cells in the epidermis, near the basal lamina, without havingthe treatment molecules pass through the skin transdermally.

[0007] U.S. Pat. No. 5,273,525 of Hofmann discloses an apparatus forelectroporation of drugs and genetic material into tissues which employsrelatively long hollow hypodermic needle for placing the drugs andgenetic material in the vicinity of the tissues to be electroporated.Whenever a hollow hypodermic is employed in a tissue, the tissue is cutwith a circular cut by the hollow hypodermic needle. As a result, when apatient receives hypodermic injection, the patient has considerablepain. To avoid such a circular cut, and to avoid the considerable paininvolved, it would be desirable if a method for delivering molecules tobiological cells were provided which does not employ a hypodermicneedle.

[0008] U.S. Pat. No. 5,318,514 of Hofmann discloses an applicator forthe electroporation of drugs and genes into cells. The applicatorincludes a plurality of needle electrodes which can be penetrated intothe skin of a patient. Material to be electroporated into the skin isretained in a fluid reservoir which wets an open cell foam elastomercarrier for the fluid. Because the material to be electroporated isretained in a fluid, in both the reservoir and the open cell foamelastomer, careful control of the amount of the material at theelectrode surfaces is difficult. It is difficult to control how muchfluid flows down from the reservoir and the open cell foam elastomer tothe surfaces of the needle electrodes, and, thereby, it is difficult tocontrol how much of the treatment molecules is actually present on thesurfaces of the electrodes 16 as the electroporation process is beingcarried out on the patient. Moreover, the presence of the fluid mediumcan have a flushing or washing effect on the tissues that areelectroporated in such a way that the electroporation process isinterfered with. In these respects, it would be desirable if anelectroporation method for delivering molecules to biological cells wereprovided which does not employ a fluid medium that flows down onto theelectrodes as the electroporation process is being carried out on thepatient.

[0009] Other disclosures relating to the use of electroporation tomediate gene transfer into epidermal cells are found in an article byReiss et al entitled “DNA-mediated gene transfer into epidermal cellsusing electroporation” in Biochem. Biophys. Res. Commun., Vol. 137, No.1, (1986), pages 244-249 and in an article by Titomirov entitled “Invivo electroporation and stable transformation of skin cells of newbornmice by plasmid DNA” in Biochim. Biophys. Acta., Vol. 1088, No. 1,(1991), pages 131-134.

[0010] U.S. Pat. No. 5,389,069 of Weaver discloses a method andapparatus for in vivo electroporation of tissues which employs arelatively long hollow cylindrical needle for providing treatingsubstances deep into tissues. As mentioned above, avoiding the usehollow cylindrical needles would be desirable to avoid the pain involvedtherewith.

[0011] U.S. Pat. Nos. 5,580,859 and 5,589,466, both of Felgner et al,disclose a method of delivering macromolecules into muscles and skin ofa patient by an injection method. Their method does not employelectroporation.

[0012] U.S. Pat. No. 5,697,901 of Eriksson discloses gene delivery intotissues by the use of a gene-carrying fluid medium that is pressurizedin conjunction with hollow microneedles. Problems of control andflushing using fluid media have been discussed hereinabove. Anelectroporation step is not employed in the Eriksson patent. As a matterof interest, Eriksson addresses the subject of pain in two respects.There is a statement that the hollow microneedle system can be used fortreating pain. There is a statement that pain in wounds can be relievedby cooling. It is noted by the present inventors herein that Erikssondoes not discuss his treatment method per se as being of a pain free orreduced pain treatment method. The present inventors theorize that thepressurized fluid injection method that is employed by Eriksson is notconducive to a pain free or reduced pain treatment method. In thisrespect, it would be desirable to provide a gene therapy treatmentmethod that employs micro-sized needles, but that does not employ apressurized fluid injection step for injecting fluid into a patient.

[0013] In an article by Henry et al entitled “MicrofabricatedMicroneedles: A Novel Approach to Transdermal Drug Delivery” in Journalof Pharmaceutical Sciences, Vol. 87, No. 8, August 1998, pages 922-925,there is a disclosure that an array of microneedles are employed topenetrate the epidermis to leave micro-sized perforations to facilitatetransdermal permeability of fluid-carried treatment agents into themicroperforated epidermis. Because the microneedles are inserted only amicroscopic distance into the epidermis, use of the microneedles ispotentially nonpainful. There is no disclosure that the microneedles areto be used as electrodes. Also, an electroporation step is not disclosedin the Henry et al article.

[0014] The following U.S. patents may be of interest for theirdisclosure of the use of relatively long electrodes in treatingbiological cells: U.S. Pat. No. 5,439,440 of Hofmann; U.S. Pat. No.5,468,223 of Mir; U.S. Pat. No. 5,674,267 of Mir et al; U.S. Pat. No.5,702,359 of Hofmann et al; U.S. Pat. No. 5,810,762 of Hofmann; and U.S.Pat. No. 5,873,849 of Bernard. It is noted that none of the patentslisted in this paragraph disclose the use of relatively long electrodeswhich have fixed electrode surfaces which are coated with a static layerof electrode releasable molecules for treating the biological cellseither when an electric field is applied to the electrodes or when thestatic layer dissolves off of the electrodes in a solvent near thebiological cells.

[0015] Further with respect to the issue of reduced pain treatment, itis noted that two important electrical parameters in electroporation areclosely related to a perceived pain in vivo. One parameter is absolutevoltage experienced by the in vivo tissue. Another parameter is thepulse width experienced by the in vivo tissue. In these respects, itwould be desirable to provide an electroporation method for deliveringmolecules to biological cells which applies relatively low absolutevoltage to cells undergoing electroporation and which can be used, ifdesired, to apply pulses having relatively short pulse width to thecells undergoing electroporation.

[0016] Still other features would be desirable in a method and apparatusfor delivery of macromolecules into epidermal cells. For example, whenelectrodes are penetrated into the epidermis, the conductive baseelectrode portions and the conductive tips of the electrodes may exhibitelectrical characteristics which are undesirable with respect to theelectroporation process in general and the biological cells that aretreated in particular. In this respect, it would be desirable if amethod and apparatus for delivery of macromolecules into epidermal cellswere provided which render nonconductive the base portions and tipportions of the electrodes.

[0017] Once electrode assemblies having a plurality of needle electrodeshave been employed on a patient, it may be a difficult task to clean andsterilize them for a subsequent use. In this respect, it would bedesirable if a method and apparatus for delivery of macromolecules intocells were provided in which the electrode assemblies are disposable.

[0018] When disposable electrode assemblies are employed, it would bedesirable if the disposable electrode assemblies are packaged in sterilepackaging.

[0019] Thus, while the foregoing body of prior art indicates it to bewell known to use electroporation to deliver molecules to biologicalcells, the prior art described above does not teach or suggest a methodand apparatus for delivery of macromolecules into cells which has mostof the following combination of desirable features: (1) does not causeskin damage that results in scarring; (2) does not leave a residue ofballistic particles in cells that are treated; (3) provides anelectroporation method for delivering molecules to biological cells inthe epidermis, near the basal lamina, without having the treatmentmolecules pass through the skin transdermally; (4) does not employ ahypodermic needle; (5) does not employ a fluid medium that flows downonto the electrodes as the electroporation process is being carried outon the patient; (6) does not employ a pressurized fluid injection stepfor injecting fluid into a patient; (7) applies relatively low absolutevoltage to cells undergoing electroporation; (8) if desired, can be usedto apply pulses having relatively short pulse width to the cellsundergoing electroporation; (9) renders the base portions and tipportions of the electrodes nonconductive; (10) provides disposableelectrode assemblies; (11) provides electrode assemblies which arepackaged in sterile packaging: and (12) permits treatment of tissuesusing coated long electrodes which have electrode releasable materialwhich includes a tissue treating agent. The foregoing desiredcharacteristics are provided by the unique electrodes coated withtreating agent and uses thereof, of the present invention as will bemade apparent from the following description thereof. Other advantagesof the present invention over the prior art also will be renderedevident.

DISCLOSURE OF INVENTION

[0020] It is noted that aspects of the invention have been disclosed incopending PCT International Application Number PCT/US00/00014, filed 12Jan. 2000, which is based upon copending U.S. Provisional ApplicationSerial No. 60/117,755, filed 28 Jan. 1999. The PCT InternationalApplication Number PCT/US00/00014 was published on 3 Aug. 2000 with PCTInternational Publication Number WO 00/44438. In addition to currentlydisclosing some of those aspects of the invention previously disclosedin the above-mentioned PCT and U.S. Provisional applications, thepresent application discloses additional invention aspects.

[0021] In accordance with one aspect of the invention, a method isprovided for delivery of molecules into biological cells which includesthe steps of:

[0022] (a) providing electrodes in an electrode assembly, wherein theelectrodes have a fixed electrode surface,

[0023] (b) coating the fixed electrode surfaces of the electrodes withat least one static layer of electrode releasable molecules to bedelivered,

[0024] (c) attaching the electrode assembly having the statically coatedelectrodes to an electrode assembly holder,

[0025] (d) providing a waveform generator for generating electricfields,

[0026] (e) establishing electrically conductive pathways between theelectrodes and the waveform generator,

[0027] (f) locating the electrodes such that the biological cells aresituated therebetween, and

[0028] (g) providing electric fields in the form of pulse waveforms fromthe waveform generator to the electrodes, such that molecules in the atleast one static layer of the electrode releasable molecules on theelectrodes are delivered into the biological cells.

[0029] On the one hand, when the static layer of electrode releasablemolecules does not include solvent separable material, thensubstantially all of the static layer of electrode releasable moleculesare electric field separable molecules. In such a case, the electricfield separable molecules are both driven off of the electrodes anddelivered into the biological cells by the applied electric fields.

[0030] On the other hand, when the static layer of electrode releasablemolecules does include solvent separable material, such as solventseparable solid material, then the static layer of electrode releasablemolecules includes both solvent separable solid material and electricfield separable molecules. In such a case, a solvent dissolves thesolvent separable material thereby releasing the electric fieldseparable molecules from the electrode, and the electric field separablemolecules are delivered into the biological cells by the appliedelectric fields. The solvent includes body fluids which are present inbody tissues.

[0031] Often, the electrode releasable molecules are in a form of astatic coating on the fixed electrode surface. In this respect, the term“static” means that the coating remains stationary on the fixedelectrode surface when either not in tissues or not under the influenceof an electric field. However, such a static coating moves off of thefixed electrode surface either when it is dissolved off of the fixedelectrode surface or when it is driven off of the fixed electrodesurface under the influence of either a solvent or a suitable electricfield, respectively.

[0032] A number of benefits can be realized by employing the staticcoated electrodes of the present invention. For example, a pre-measuredquantity of a static layer of electrode releasable molecules can beretained on the fixed electrode surfaces. Such a pre-measured quantityof the static layer of electrode releasable molecules can serve as apre-measured dose of material to be delivered to the biological cells.Moreover, the static coated electrodes can be coated with a concentratedquantity of the electrode releasable molecules. In addition, the staticcoated electrodes can be pre-packaged so that when they are removed fromtheir package, they are rapidly ready for use, without the need forconventional preparatory steps such as dilution and hypodermicinjection.

[0033] Also, in accordance with aspects of the present invention, anelectrode includes an electrode underbody and a fixed electrode surfacewhich lies on top of the electrode underbody. The fixed electrodesurface can be implemented in a wide variety of embodiments. Forexample, most simply, the simple surface of the electrode itself canserve as the fixed electrode surface which lies on top of the electrodeunderbody. The fixed electrode surface can be a smooth electrodesurface, can be an oxidized metal surface (e. g. oxides of silver,nickel, and copper), can include fixed metal particles, and can be aroughened surface.

[0034] Also, in accordance with aspects of the present invention, theelectrode releasable material on the fixed electrode surface can be in aform of a gel coating, a solid layer of nonpolymeric material, and apolymer layer.

[0035] Varieties of the fixed electrode surface and the static,electrode releasable material can be mixed and matched.

[0036] Some specific examples of combinations of the fixed electrodesurface and the electrode releasable material include: a fixed electrodesurface having metal oxides and electrode releasable material includingDNA; a fixed electrode surface being a smooth surface and the electrodereleasable material in the form of a solid coating; a fixed electrodesurface having an etched rough surface and the electrode releasablematerial as either a solid nonpolymeric layer, a gel layer, or apolymeric layer.

[0037] The pulse waveforms may be provided by applying a sequence of atleast three single, operator-controlled, independently programmed, DCelectrical pulses, to the biological cells. The sequence of at leastthree DC electrical pulses has one, two, or three of the followingcharacteristics (a) at least two of the at least three pulses differfrom each other in pulse amplitude, (b) at least two of the at leastthree pulses differ from each other in pulse width, and (c) a firstpulse interval for a first set of two of the at least three pulses isdifferent from a second pulse interval for a second set of two of the atleast three pulses.

[0038] Additionally, the method can include a step of providing theelectrode assembly holder with electrically conductive pathways betweenthe electrode assembly and the waveform generator.

[0039] In addition, the method can include a step of providing theelectrode assembly in a sterile package. In such a case, the electrodeassembly is removed from the sterile package prior to use.

[0040] Further, the method can include the steps of providing theelectrodes with electrically insulated outer surface electrode tipportions and electrically insulated outer surface electrode baseportions.

[0041] The molecules in the at least one static layer of molecules inthe electrode coating preferably include macromolecules. Themacromolecules in the electrode coating can include a tissue treatingagent, a polynucleotide vaccine (DNA vaccine and/or RNA vaccine), or aprotein-based vaccine, among others.

[0042] With a variation of the method of the invention, the moleculescan be delivered to Langerhans cells in epidermal tissue of a patientwith reduced sensation (reduced pain or nearly painless or pain free) tothe patient. To provide reduced sensation delivery of molecules to thepatient, the following conditions are maintained (a) the pulse waveformshave an absolute applied voltage in a range of 0.1 to 300 volts; (b) theelectrodes of opposite polarity are separated by a separation distancein a range of from 50 to 500 microns; and (c) the electrodes arepenetrated into the epidermal tissue a distance up to and slightlybeyond the basal lamina layer of the epidermal tissue.

[0043] With another variation of the method of the invention, themolecules can be delivered to a tissue which is deeply located underhealthy tissue. With such a variation of the method of the invention,the electrodes are long enough to penetrate through the healthy tissueand into the tumor. The fixed electrode surface portions of theelectrodes that penetrate the tumor are coated with electrode releasablematerial that includes a deep tumor tissue treating agent.

[0044] The pulse waveforms which drive the molecules of the electrodereleasable coating molecules off of the electrodes are electrophoresiswaveforms. The pulse waveforms which deliver the driven-off moleculesinto the biological cells are electroporation waveforms. For a staticlayer of electric field separable molecules, common pulse waveforms bothdrive the coating molecules off of the electrodes and deliver thedriven-off molecules into the biological cells.

[0045] The biological cells can be in vivo, ex vivo, or in vitro. Morespecifically, the biological cells can be in epidermal tissue and can beLangerhans cells in the epidermal tissue. Also, the biological cells canbe in deep tissues, and can be in tumors in deep tissues.

[0046] In accordance with another aspect of the invention, an apparatusis provided for delivery of molecules into biological cells and includesa waveform generator which provides pulse waveforms. An electrodeassembly holder is provided, and an electrode assembly is mechanicallysupported by the electrode assembly holder. The electrode assemblyholder is also electrically connected to the waveform generator throughelectrically conductive pathways. The electrode assembly includeselectrodes which are coated with at least one static layer of moleculesto be delivered into the biological cells.

[0047] The electrode assembly can be removable and replaceable from theelectrode assembly holder. In this respect, the electrode assemblyincludes electrode-assembly-conductive strips. The electrode assemblyholder includes holder conductors which are registrable with theelectrode-assembly-conductive strips when the electrode assembly ismechanically connected to the electrode assembly holder. Also, theelectrode assembly holder includes electrically conductive pathwaysbetween the holder conductors and the waveform generator.

[0048] With the apparatus, a sterile package can be provided for theelectrode assembly. The sterile package is removed from the electrodeassembly after the electrode assembly is mechanically supported by theelectrode assembly holder and is electrically connected to the waveformgenerator.

[0049] With the apparatus, if desired, the waveform generator providespulse waveforms which include a sequence of at least three single,operator-controlled, independently programmed, DC electrical pulses, tothe biological cells. The sequence of at least three DC electricalpulses has one, two, or three of the following characteristics (a) atleast two of the at least three pulses differ from each other in pulseamplitude, (b) at least two of the at least three pulses differ fromeach other in pulse width, and (c) a first pulse interval for a firstset of two of the at least three pulses is different from a second pulseinterval for a second set of two of the at least three pulses.

[0050] The electrodes can include electrically insulated outer surfaceelectrode tip portions and electrically insulated outer surfaceelectrode base portions. The electrodes are coated with at least onestatic layer of molecules, which may include macromolecules, which mayinclude a tissue treating agent, a polynucleotide vaccine (a DNA vaccineand/or a RNA vaccine) and/or a protein-based vaccine.

[0051] The static layer of electrode releasable macromolecules on theelectrodes (e. g. a tissue treating agent, a polynucleotide vaccine, ora protein-based vaccine, among others) can be in a variety of formsprior to using the electrodes on a patient. More specifically, thestatic layer of macromolecules can be in a solid form, coating the solidelectrodes. Also, the static layer of macromolecules can be in a gelform or can be in a form of a liquid fixed on a fixed surface matrix ofthe electrodes. The fixed surface matrix can be solid surface particles(e. g. metal particles), a liposome matrix, or a solid polymer matrix.

[0052] In accordance with yet another aspect of the invention, apackaged sterile electrode assembly is provided which includes a sterileelectrode assembly which includes electrodes which are coated with atleast one static layer of molecules to be delivered into biologicalcells. The electrode assembly includes electrode-assembly-conductivestrips for connection to complementary electrically conductive pathwaysleading to the waveform generator. In addition, an internally sterilepackage encloses the sterile electrode assembly contained therein.

[0053] With the packaged sterile electrode assembly, the electrodes caninclude electrically insulated outer surface electrode tip portions andelectrically insulated outer surface electrode base portions.

[0054] With the packaged sterile electrode assembly, the electrodes arecoated with macromolecules which can include a solid phasepolynucleotide (DNA vaccine and/or RNA vaccine) and/or a solid phaseprotein-based vaccine. Also, the polynucleotide vaccine or protein-basedvaccine can be in a gel form or can be in a form of a liquid fixed on afixed surface matrix of the electrodes, prior to using the electrodes ona patient. The fixed surface matrix can be solid surface particles (e.g. metal particles), a liposome matrix, or a solid polymer matrix.

[0055] In accordance with the invention, transfection of cells with DNAin vivo, using electric field mediated transfection, is an efficientprocess. Additionally, electric fields can be used for the delivery ofother macromolecules such as RNA and proteins into cells. In the priorart, the electric field delivery has one disadvantage, that being thepain induced by the high voltage electrical pulses required for thetransfection. In contrast, as described herein, a method is provided fordelivering macromolecules (DNA, RNA, and protein) to cells, in tissuesin vivo, using painless (or nearly painless) and efficient electricfield mediated delivery.

[0056] A number of applications of the method and apparatus for deliveryof macromolecules into cells, of the invention, are contemplated.Briefly, such applications include treating organ tissues,polynucleotide vaccination, protein vaccination, and gene therapy.

[0057] For treating deep tumor tissues, it is important to maximizedelivery of the deep tumor tissue treating agent to the tumor tissue andto minimize deliver of the agent to healthy tissue.

[0058] For DNA vaccination, there are two overriding requirements. Oneis gene expression in vivo and the other is that antigen-presentingcells must either obtain antigen from a nearby, transfected cell orexpress the antigen themselves. The highest concentration of accessibleantigen presenting cells resides in the skin as cells called Langerhanscells. These cells are part of a very effective group of antigenpresenting cells called dendritic cells. Electroporation is a viablealternative method for transfecting selected cells in vivo.

[0059] Proteins also can be introduced into cells using electric fieldmediated delivery. In conventional vaccination, proteins are deliveredoutside cells using a hypodermic needle. This type of delivery isinefficient in inducing a cell mediated cytotoxic lymphocyte immuneresponse. Some infectious diseases require a cytotoxic lymphocyteresponse as a component of the immune response for efficient clearanceof the infection. Delivery of proteins into cells promotes the inductionof that response.

[0060] Delivery of therapeutic genetic medicine into cells for thepurpose of making those cells express a missing protein is the basis ofgene therapy. Using relatively short electrodes of the invention, themethod and apparatus of the invention can be used to deliver therapeuticDNA into cells on the surface of any accessible organ in addition to theskin. Using relatively long electrodes of the invention, the method andapparatus of the invention can be used to deliver therapeutic DNA intocells deep into tissues and organs.

[0061] The method of the invention can be used in a method for painless,effective delivery of macromolecules to epidermal tissues, in vivo, forthe purpose of vaccination (or treatment), DNA vaccination, genetherapy, or other reasons.

[0062] An electrode with at least one of two characteristics is used fordelivery of macromolecules into cells in epidermal tissue. One of thetwo characteristics is an electrode length short enough that it does notpenetrate to a depth in tissue with nerve endings. Anothercharacteristic is that inter-electrode distances are small enough toallow pulse parameters (voltage and pulse width) to be used that arepainless. Only one or the other of these characteristics is needed inany given epidermal application, however, they may be used together.

[0063] The above brief description sets forth rather broadly the moreimportant features of the present invention in order that the detaileddescription thereof that follows may be better understood, and in orderthat the present contributions to the art may be better appreciated.There are, of course, additional features of the invention that will bedescribed hereinafter and which will be for the subject matter of theclaims appended hereto.

[0064] In this respect, before explaining preferred embodiments of theinvention in detail, it is understood that the invention is not limitedin its application to the details of the construction and to thearrangements of the components set forth in the following description orillustrated in the drawings. The invention is capable of otherembodiments and of being practiced and carried out in various ways.Also, it is to be understood, that the phraseology and terminologyemployed herein are for the purpose of description and should not beregarded as limiting.

[0065] As such, those skilled in the art will appreciate that theconception, upon which disclosure is based, may readily be utilized as abasis for designing other structures, methods, and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe present invention.

[0066] In view of the above, it is an object of the present invention toprovide a new and improved method and apparatus for delivery ofmacromolecules into cells which does not cause skin damage that resultsin scarring.

[0067] Another object of the present invention is to provide a new andimproved method and apparatus for delivery of macromolecules into cellswhich does not leave a residue of ballistic particles in cells that aretreated.

[0068] Even another object of the present invention is to provide a newand improved method and apparatus for delivery of macromolecules intocells that provides an electroporation method for delivering moleculesto biological cells in the epidermis, near the basal lamina, withouthaving the treatment molecules pass through the skin transdermally.

[0069] Still a further object of the present invention is to provide anew and improved method and apparatus for delivery of macromoleculesinto cells which does not employ a hypodermic needle.

[0070] Yet another object of the present invention is to provide a newand improved method and apparatus for delivery of macromolecules intocells that does not employ a fluid medium that flows down onto theelectrodes as the electroporation process is being carried out on thepatient.

[0071] Still another object of the present invention is to provide a newand improved method and apparatus for delivery of macromolecules intocells which does not employ a pressurized fluid injection step forinjecting fluid into a patient.

[0072] Yet another object of the present invention is to provide a newand improved method and apparatus for delivery of macromolecules intocells that applies relatively low absolute voltage to cells undergoingelectroporation.

[0073] Still a further object of the present invention is to provide anew and improved method and apparatus for delivery of macromoleculesinto cells that can be used, if desired, to apply pulses havingrelatively short pulse width to the cells undergoing electroporation.

[0074] Yet another object of the present invention is to provide a newand improved method and apparatus for delivery of macromolecules intocells which renders the base portions and tip portions of the electrodesnonconductive.

[0075] Still a further object of the present invention is to provide anew and improved method and apparatus for delivery of macromoleculesinto cells that provides disposable electrode assemblies.

[0076] Yet another object of the present invention is to provide a newand improved method and apparatus for delivery of macromolecules intocells which electrode assemblies are packaged in sterile packaging.

[0077] Still another object of the present invention is to provide a newand improved method and apparatus for delivery of macromolecules intocells which permit treatment of tissues using coated long electrodeswhich have electric field separable material which includes a tissuetreating agent.

[0078] These together with still other objects of the invention, alongwith the various features of novelty which characterize the invention,are pointed out with particularity in the claims annexed to and forminga part of this disclosure. For a better understanding of the invention,its operating advantages and the specific objects attained by its uses,reference should be had to the accompanying drawings and descriptivematter in which there are illustrated preferred embodiments of theinvention.

BRIEF DESCRIPTION OF DRAWINGS

[0079] The invention will be better understood and the above objects aswell as objects other than those set forth above will become moreapparent after a study of the following detailed description thereof.Such description makes reference to the annexed drawing wherein:

[0080]FIG. 1 is a schematic illustration of the overall apparatus of theinvention.

[0081]FIG. 2 is a schematic illustration of relatively short electrodescoated with a static layer of macromolecules, with the electrodespenetrating an epidermal skin layer, and with the macromolecules beingdriven by pulse waveforms off of the electrodes to delivermacromolecules into epidermal cells.

[0082]FIG. 3 is a schematic illustration of tip portions of theelectrodes.

[0083]FIG. 4 is a schematic illustration of relatively long electrodescoated with a static layer of macromolecules, with the electrodespenetrating through healthy tissue into the tissue of a tumor.

[0084]FIG. 5 schematically shows an electrode which has a fixedelectrode surface that is coated with a static layer of electrodereleasable molecules.

[0085]FIG. 6 schematically shows apparatus used for coating theelectrodes with macromolecules.

MODES FOR CARRYING OUT THE INVENTION

[0086] A method and apparatus are provided for delivery ofmacromolecules into cells, and with reference to the drawings, saidmethod and apparatus are described below. The method for delivery ofmolecules into biological cells employs the apparatus set forth andincludes the steps of

[0087] (a) providing electrodes 16 in an electrode assembly 12, whereinthe electrodes have a fixed electrode surface 42,

[0088] (b) coating the fixed electrode surfaces 42 of the electrodes 16with at least one static layer of electrode releasable molecules 44 tobe delivered,

[0089] (c) attaching the electrode assembly 12 having the staticallycoated electrodes 16 to an electrode assembly holder 13,

[0090] (d) providing a waveform generator 15 for generating electricfields,

[0091] (e) establishing electrically conductive pathways between theelectrodes 16 and the waveform generator 15,

[0092] (f) locating the electrodes 16 such that the biological cells aresituated therebetween, and

[0093] (g) providing electric fields in the form of pulse waveforms fromthe waveform generator 15 to the electrodes 16, such that molecules inthe at least one static layer of the electrode releasable molecules 44on the electrodes 16 are delivered into the biological cells.

[0094] Referring to FIG. 5, an electrode 16 includes an electrodeunderbody 40 and a fixed electrode surface 42 which lies on top of theelectrode underbody 40. A static layer of electrode releasable molecules44 is located on top of the fixed electrode surface 42. It is noted,however, that in different embodiments, the fixed electrode surface 42and the static layer of electrode releasable molecules 44 can beintermingled.

[0095] In some embodiments of the invention, the static layer ofelectrode releasable molecules 44 is homogeneous and includessubstantially only electric field separable molecules. In otherembodiments, the electrode releasable molecules 44 include only asolvent separable material. In still other embodiments, the electrodereleasable molecules 44 include both electric field separable moleculesand solvent separable material. Generally, the solvent separablematerial is in a solid state. Alternatively, the solvent separablematerial can be in a gel state.

[0096] With reference to FIG. 4, in one variation of the method, usinglong needle electrodes 34 which are coated with a static layer ofelectrode releasable molecules 44, e. g. macromolecules 18 which includea deep tumor tissue treating agent, a deep tumor tissue 38 is treatedwith the macromolecules 18 which are driven off of the long needleelectrodes 34 by an imposed electric field.

[0097] With reference to FIG. 4, a specific example of the aboveprocedure is the use of plasmids expressing lymphokines for cancerimmunotherapy. In one example, plasmids expressing interleukin-2 arecoated onto the long needles 34 using the described method. Morespecifically, the macromolecules 18 in FIG. 4 include the plasmidsexpressing interleukin-2. The long needles 34 are then inserted throughhealthy tissue 33 into selected tumor tissue 38. Then, theelectrophoretic and electroporation pulses administered. This method isused to induce a local immune response to the tumor tissue 38 in aneffort to generate a generalized immune response. Plasmids expressingother lymphokines can be used in this procedure. Examples areinterleukin-12, interleukin-4, interferon-gamma and a variety of others.Plasmids expressing other immune system molecules such as co-stimulatorymolecules or adhesion molecules can also be used.

[0098] In another variation of the method, referring to FIG. 2, thestatically-coated molecules are delivered with reduced sensation in apatient to Langerhans cells 22 in the epidermis 20 of a patient. Thepulse waveforms have an absolute applied voltage in a range of from 0.1to 300 volts. Electrodes 16 of opposite polarity are separated by aseparation distance in a range of from 50 to 500 microns. Thestatically-coated electrodes 16 are penetrated into the epidermal tissueup to and slightly beyond the basal lamina layer of the epidermaltissue.

[0099] The pulse waveforms which drive the coating molecules off of theelectrodes 16 are electrophoresis waveforms. The electrophoresiswaveforms can be in a range of from 0.1 to 100 volts/cm. The pulsewaveforms which deliver the driven-off molecules into the biologicalcells are electroporation waveforms. The electroporation waveforms canbe in a range of from 100 to 10,000 volts/cm. Common pulse waveformsboth drive the coating molecules off of the electrodes 16 and deliverthe driven-off molecules into the biological cells.

[0100] The biological cells to which the molecules are delivered can bein vivo, ex vivo, or in vitro. More specifically, as shown in FIG. 2,the biological cells can be in the epidermis 20 (epidermal tissue) andcan be Langerhans cells 22 in the epidermal tissue.

[0101] The statically-coated molecules driven off of the electrodes 16by electrophoresis electrical pulses are delivered to the cells byelectroporation pulses. In accordance with an exemplary protocol, thepulse waveforms are provided by the waveform generator 15 by applying asequence of at least three single, operator-controlled, independentlyprogrammed, DC electrical pulses, to the biological cells. The sequenceof at least three DC electrical pulses has one, two, or three of thefollowing characteristics (1) at least two of the at least three pulsesdiffer from each other in pulse amplitude, (2) at least two of the atleast three pulses differ from each other in pulse width, and (3) afirst pulse interval for a first set of two of the at least three pulsesis different from a second pulse interval for a second set of two of theat least three pulses.

[0102] The electrode assembly holder 13 is provided with electricallyconductive pathways, which includes conductors 21, between the electrodeassembly 12 and the waveform generator 15.

[0103] The electrode assembly 12 can be provided in a sterile package 24which is removed from the electrode assembly 12 prior to use.

[0104] Preferably, for electrodes 16 used with epidermal tissue, theelectrodes 16 have conical tips, whereby they are referred to as needleelectrodes. The electrodes 16 can be provided with electricallyinsulated outer surface electrode tip portions 17 and electricallyinsulated outer surface electrode base portions 19. The electricallyinsulated outer surface electrode base portions 19 minimize currentflowing across the upper skin surface when the pulse voltage is applied.Moreover, DNA binds poorly to the electrically insulated outer surfaceelectrode base portions 19. The outer surface area of the electrodes 16between the electrically insulated outer surface electrode tip portions17 and the electrically insulated outer surface electrode base portions19 does not have an outer insulation layer and is a good surface for DNAbinding. The outer surface areas between the electrically insulatedouter surface electrode tip portions 17 and the electrically insulatedouter surface electrode base portions 19 is conductive and can bereferred to as an active electrode area. The electrically insulatedouter surface electrode tip portions 17 prevent large local electricfield intensity which may cause burning in the tissue.

[0105] An electrode assembly 12 that is suitable for delivering DNAvaccines to Langerhans cells 22 in the epidermis 20 of the forearm hasthe following characteristics:

[0106] (a) electrode length—130 microns

[0107] (b) electrode material resistivity—less than 0.1 ohm-cm

[0108] (c) insulation at tip—extending upward 10 microns from tip end

[0109] (d) insulation at base—extending downward 55 microns fromelectrode carrier

[0110] (e) electrode tip flatness—less than 1 square micron

[0111] (f) electrode diameter at base—43 microns

[0112] (g) electrode spacing in a conductive row—130 microns

[0113] (h) number of electrodes in a conductive row—35

[0114] (i) space between conductive rows—260 microns (2×130)

[0115] (j) number of conductive rows—25.

[0116] For epidermal applications, the lengths of the electrodes 16 aredetermined by the thickness of the epidermis 20. The thickness of theepidermis 20 varies in different parts of the human body. For example,the thickness of the epidermis 20 on the medial forearm or the lateralupper arm above the deltoid muscle is considerably thinner than thethickness of the epidermis 20 on the heel or sole of the foot.

[0117] Referrring to FIG. 5, molecules in the static layer of electricfield separable molecules 44 preferably include macromolecules 18. Themacromolecules 18 in the electrode coating can include a deep tumortissue treating agent, a polynucleotide vaccine (e.g a DNA vaccine or anRNA vaccine), and/or a protein-based vaccine. The polynucleotide vaccineand the protein-based vaccine can be in the form of a solid phase DNAvaccine or protein-based vaccine applied to the electrodes 16. Also, thepolynucleotide vaccine or protein-based vaccine can be in a gel form orcan be in a form of a liquid fixed on a fixed surface matrix of theelectrodes. The fixed surface matrix can be solid surface particles (e.g. metal particles), a liposome matrix, or a solid polymer matrix.

[0118] Preferably, the electrode assembly 12 is removable andreplaceable from the electrode assembly holder 13. The electrodeassembly 12 includes electrode-assembly-conductive strips. The electrodeassembly holder 13 includes holder conductors which are registrable withthe electrode-assembly-conductive strips when the electrode assembly 12is mechanically connected to the electrode assembly holder 13. Theelectrode assembly holder 13 includes electrically conductive pathwaysbetween the holder conductors and the waveform generator 15.

[0119] As stated above, there are three main components required for thedelivery of macromolecules into cells in tissue. They are a waveformgenerator 15, an electrode assembly holder 13, and a statically-coatedelectrode assembly 12. The waveform generator supplies the electricalpulses necessary for generating the electric field in the tissue. Theelectrode assembly 12 contains the electrodes 16, and the polynucleotideor protein macromolecules are applied to the electrodes 16 as a staticcoating. The electrode assembly holder 13 connects the electrodeassembly 12 to the waveform generator 15.

[0120] The statically-coated electrode assembly 12 can be in the form ofan electrode array can be in the form of a disposable, one-time-useelectrode array which has the macromolecules pre-loaded onto theelectrodes. In this respect, the pre-loaded electrode array can beprovided as a sterile package. To use such an electrode array, thesterile package is opened, and the electrode array is connected to theelectrode array holder. The electrode assembly holder is grasped by aperson, and portions of the electrode assembly are forced into theselected tissue of a patient. For epidermal tissue, preferably, the tipsof the electrodes in the statically-coated electrode assembly arelocated in the region of the Langerhans cells, which are dendritic cellsof the epidermis.

[0121] Then, a pulse waveform is sent from the waveform generator,through the electrode assembly holder, and to the statically-coatedelectrode assembly. The pulse waveform drives pre-loaded macromoleculesoff of the statically-coated electrode assembly and into the epidermis.In the epidermis, the pulse waveform electropermeabilizes the targetepidermal cells so that the macromolecules enter the target cells.

[0122] As illustrated in FIG. 2, an electrode assembly 12 includes anon-conductive electrode carrier 14 and a plurality of individual needleelectrodes 16 supported by the electrode carrier 14. The active areas 29of the electrodes 16 are statically coated with macromolecules which areillustrated as small “x's” 18 on the surfaces of the electrodes 16.Under the influence of the pulse waveforms, some of the macromolecules18 are driven off of the electrodes 16 by electrophoresis voltage andenter the epidermis 20 and are delivered to the dendritic Langerhanscells 22 and the living epithelial cells 23 in the living epidermisabove the basal lamina 25 in the epidermis 20 by electroporationvoltage.

[0123] The waveform generator 15 produces the pulses for the protocol.The output of the waveform generator can be conventional with a singleselection of pulse parameters such as voltage, pulse width, intervalbetween pulses, number of pulses and the vector of the pulse.Alternatively, the output of the waveform generator can be programmablewith the ability to change any of the parameters (voltage, pulse width,interval between pulses, number of pulses,) from pulse to pulse. Thevariable output is needed for optimal performance because a differentelectric field is required for macromolecule movement off of theelectrodes 16 than the electric field required for electric fieldmediated delivery of macromolecules into cells. A suitable programmablepulse generator is the PulseAgile (Registered in U.S. Patent andTrademark Office), Model PA-4000 Electroporation System made by CytoPulse Sciences, Inc., P. 0. Box 609, Columbia, Md. 21045. It is notedthat the Model PA-4000 delivers rectangular waves of various amplitudes(voltages), width, and intervals.

[0124] In addition to programmable control of voltage, pulse width,interval between pulses and number of pulses from pulse to pulse,programmable control of two other parameters is desired. One is controlof the direction or vector of the applied electric field. The other iscontrol of electrode selection. In one application, electric fielddirection could be reversed to insure better distribution of themacromolecule. In another application, individual pairs of electrodearrays could be sequentially selected.

[0125] A suitable device for electrode selection and the selection ofelectrode field direction is the programmable pulse switch, which is anoptional component of the above-mentioned PA-4000 ElectroporationSystem.

[0126] The statically-coated electrode assembly 12 serves two functions.It delivers the macromolecule to the desired site and it delivers theelectric field to the tissue.

[0127] The statically-coated electrode assembly 12 includes:

[0128] 1. a non-conductive electrode carrier 14.

[0129] 2. an array of needle electrodes 16 fabricated on the electrodecarrier 14, wherein the needle electrodes 16 are statically coated.

[0130] 3. Electrode-assembly-conductive strips for electrical connectionto the holder conductors on the electrode assembly holder 13 to connectelectrically to the waveform generator 15.

[0131] In carrying out the method of the invention for epidermal use,the tips of the statically-coated needle electrodes 16 are pressedagainst the epidermis 20 of a patient with the needles puncturing thestratum corneum 27 and extending into the epidermis 20 and the upperdermis 31 as shown in FIG. 2.

[0132] The electrode needles can have many shapes. Examples of needleelectrode shapes are: cylindrical needles, flat needles, cone shapedneedles, and blade needles. The needles can be pointed rounded or blunt.Each of these shapes can be single or multiple per electrode row.

[0133] The purposes of the electrode assembly holder are to establish anelectrical connection between the waveform generator and the electrodeassembly and to provide a support for the electrode assembly when theelectrode assembly is applied to the patient. It provides a mechanicalconnection for application to the patient. It also provides a means ofdelivering the electrode assembly to the patient's tissue whilemaintaining sterility of the electrode assembly.

[0134] The statically-coated electrode assembly can have the followingoptional features. It can have means to provide proper pressure on theelectrode assembly to the tissue. It can have indicators that indicatecorrect application pressure, on-going electrical delivery, andcompletion of electrical delivery. It can have a switch for initiationof the pulse protocol. It can have a means for automatically initiatinga pulse protocol when proper pressure is applied to the electrodeassembly holder.

[0135] As stated above, macromolecules, including DNA and proteinmacromolecules, need to be driven off of the statically-coated coatedelectrodes 16 by electrophoresis voltages so that they can move throughthe extra-cellular spaces of tissue prior to the application ofelectroporation pulses for delivering the macromolecules into thetargeted biological cells in the tissue.

[0136] As stated above, the macromolecules 18 in the static layer ofelectric field separable molecules 44 are initially bound to the fixedelectrode surfaces 42 of the electrodes 16. In a mechanical approach tocoating the electrodes 16 with a static coating of macromolecules 18, arelatively high concentration of macromolecules 18 is dissolved orsuspended in a solvent or liquid carrier. The electrodes 16 are thendipped into the solution or suspension. Then, the solvent or liquidcarrier is evaporated, leaving a static coating of macromolecules 18, inthis case a solid coating of macromolecules 18, on the electrodes 16.Alternatively, the electrodes 16 are coated by spraying. Othermechanical means of coating the electrodes 16 are possible.

[0137] Macromolecules such as DNA bind with good efficiency to manysurfaces. The physical and chemical properties of the material can beused to enhance binding to electrode surfaces to provide thestatically-coated electrodes 16.

[0138] Molecules tend to bind to each other through various molecularinteractions, each having a different binding strength. These sameforces are active between solid substrates and soluble molecules as wellas among molecules in solution. The molecular interactions are:

[0139] 1. Solvation: Solvent binding. An interaction between thecomponents of a molecule and the solvent molecules.

[0140] 2. Hydrophobic interaction: A solute-solute interaction as aconsequence of the inability to interact with the solvent; an avoidanceinteraction

[0141] 3. Van der Walls forces are weak attractions that exist betweenall molecules. It is effective only at short distances and can bestronger if interactions based upon complementary shape

[0142] 4. Hydrogen bonds are bonds formed between hydrogen and othermolecules such as nitrogen and oxygen.

[0143] 5. Ionic bonds are attractions based upon attraction ofoppositely charged portions of molecules.

[0144] 6. Covalent bonds are the strongest of molecular bonds.

[0145] More specifically with respect to DNA, DNA is both sparinglysoluble in water and charged. The organic rings within the nucleotidesimpart the hydrophobic properties to DNA. The phosphate molecules in theDNA polymer., impart a net negative charge.

[0146] The strongest bond between an electrode surface and DNA is thehydrophobic bond. When an electrode has a positive charge, DNA movestowards the electrode thereby enhancing the interaction of DNA with theconductive hydrophobic surface. For delivery of the DNA to biologicaltissues, the electrical charge is reversed. Migration of macromolecules18 from the electrode surface occurs as soon as the repelling force oflike charges exceeds the force of the hydrophobic and other molecularinteraction.

[0147] DNA can be coated onto specific sites by binding the DNA to metal(such as an electrode surface.) or another conductive material throughthe use of a positive charge. Subsequently, for driving the DNA off ofthe electrode surface and for subsequently delivering the DNA tobiological cells, a negative charge is applied to the same surface. DNA,being negatively charged, will migrate in an electric field toward thepositive electrode. This phenomenon is called electrophoresis. If thepositive electrode is a hydrophobic surface as are most metals, thepositive charge and the hydrophobic interaction will work together tohold the DNA to the surface.

[0148] Most macromolecules have a net charge in solution at a pH otherthan its iso-electric point. DNA, for instance, is negatively charged atphysiological pH. This means that a DNA molecule will migrate towards apositive electrode. This property is used to bring the macromolecule incontact with the electrode where binding of the DNA to the electrodesurfaces occurs via the other molecular interactions listed. DNA, forinstance, can bind because it is hydrophobic.

[0149] Electrical coating takes advantage of the charge of themacromolecules. A stated above, DNA is negatively charged and thereforemigrates to a positively charged electrode. Reference is made to FIG. 6which illustrates an apparatus used for coating the electrodes 16 with astatic coating. In one coating process, DNA is added to a buffersolution and then placed into a chamber with an electrode that serves asthe cathode. Preferably this electrode is separated from the buffer by agel interface to prevent metal of the cathode coming into contact withthe DNA. The electrode device is inserted into the liquid, and apositive charge is applied to the electrode device drawing the DNA tothe surface of the electrode device. The DNA attaches to the surface ofthe electrode device by hydrophobic or other interaction to provide astatically-coated electrode until the DNA is expelled by a reversecharge. The DNA is dried on to the device with or without a protectant,such as sugars, and with or without other carrier molecules. Substancescan also be added to the static coating on the electrodes which promoteuptake of the treating material into the target cells.

[0150] The amount of statically-coated macromolecule on the electrodeassembly varies depending upon the application. For DNA immunization,for example, the electrode assembly is loaded with 0.01 to 100micrograms of plasmid DNA.

[0151] Sterile materials and a sterile local environment can be used inthe manufacture of the electrode assembly with the macromolecule.Alternatively, the assembly can be sterilized after manufacture.

[0152] A typical sequence of steps in administering macromolecules 18 toa patient using the method and apparatus of the invention are describedas follows. In a clinic, the waveform generator 15 would be connected tothe electrode assembly holder 13. For an individual application, anelectrode assembly 12 whose electrodes 16 have been loaded with a staticcoating of the desired macromolecule is selected. The electrode assembly12 is then mechanically connected to the electrode assembly holder 13.As the electrode assembly holder 13 is grasped by an operator, thestatically-coated electrodes 16 are pressed onto the patient's tissue(e. g. skin). For skin, the statically-coated electrodes 16 penetrateinto the epidermis 20 and extend substantially only to the basal laminalayer. After the statically-coated electrodes 16 have been locatedthusly in the epidermis 20, the macromolecular delivery process isstarted, and the selected pattern of electric fields is initiated. Aftercompletion of the delivery protocol, the electrodes 16 are removed fromthe epidermis 20, and the electrode assembly 12 is discarded.

[0153] As stated above, when a solvent separable solid material is notemployed in the electrode releasable molecules 44, the electricalprotocol is designed to drive the statically-coated macromolecules offof the electrodes 16 into the tissue, followed by delivery of themacromolecules into cells in the tissue. For DNA, a typical sequence ofelectrical pulses is as follows. First, a series of low voltage(electrophoresis) pulses are applied to the electrodes 16 to remove theDNA from all negatively charged electrodes. Typically, alternating rowsof electrodes are negatively charged. Next, higher voltageelectroporation pulses are applied to the electrodes 16 to drive the DNAinto cells. Next, electrode polarity is reversed and low voltage pulsesdelivered with opposite polarity to remove the DNA from the remainingelectrodes. Higher voltage electroporation pulses are then applied toforce the DNA into cells.

[0154] A significant use of this macromolecular delivery system is todeliver macromolecules to skin. For this use, electrode needle length ischosen to allow penetration of the electrode to the stratum basalis andbasement membrane (basal lamina). Some slight penetration into thedermis may occur. For this use on a patient's arm, an electrode lengthof 130 microns is selected. This depth allows treatment of cells of theepidermis. For a DNA vaccine or gene therapy, the cells transfected bythis delivery method are dendritic cells (skin Langerhans cells 22) andepithelial cells.

[0155] Aside from administering macromolecules to biological cells inthe epidermis, the method and apparatus of the invention can be used inother biological environments, such as tissues during surgery and withplants.

[0156] A wide variety of methods can be employed for manufacturing theelectrode assembly 12 of the invention, prior to application of thestatic coating. A number of examples are presented below.

[0157] Standard microchip manufacturing processes can be adapted to makethe conductive microneedles on a non-conductive support, as inaccordance with the invention. In one example, a blank consisting of asilicon or other non conductive layer and a metal layer would be used.The mask would be designed to encourage more etching between rows thanwithin rows, resulting in conductive rows of electrodes withnonconductive spaces between rows.

[0158] Another method of construction of an electrode assembly is byadapting the known technique of extrusion micro-fabrication, and anexample follows. Electrode material and adjacent insulating material areprepared by mixing a ceramic, metal or other powder with a thermoplasticbinder. The individual components are assembled and warm pressed tostick together. The resulting rod is extruded to reduce its size.Following the extrusion, the new rods are assembled in a rod composed ofa multiple of the extruded rod. This newest rod is re-extruded to reducethe size of the multiple rods to the size of the first extruded rod.After the size is reduced to the desired size the parts can be heated toremove the binder. A second, higher heat is used to sinter the metal orceramic powders together. The rods are cut into disks before or afterthe sintering. Differential sand blasting or other mechanical orchemical techniques can be used to raise the needles above the surfaceof the insulator.

[0159] Another method for manufacture would be to use laser millingtechniques to remove material from a sandwich composed of conductive andnonconductive layers.

[0160] For some of the arrays of electrodes, the distance between theelectrodes is large enough for mechanical assembly. An example of suchassembly follows. Wire of the desired metal composition and diameter isarranged on spools for assembly. The wires are fed into an apparatusthat aligns the wire to the correct distance apart. Ceramic or plasticmaterial is injected into a flow through system that results in completefilling of the gap between the electrodes and forms the shape of theoutside rim of the electrode. The plastic or ceramic is hardened and cutinto discs. The resulting disks are differentially eroded, takingadvantage of the softer matrix. The erosion can be done using mechanicalmethods, chemical methods or a combination of methods. The surfaceerosion leaves needles of the desired length protruding above thesupporting matrix.

[0161] Another manufacturing technique for an electrode assembly isdescribed as follows. Stainless steel needles 30 mm in length and 120microns in diameter are obtained. One source is from an acupuncturesupply company. Seirin No. 02 needles are an example. The needles arecut from the handle if one is present. A number of needles are selectedfor each row of the device. Thirty-five needles per row are used forthis example. The needles are carefully placed side by side with thetips of the needles in line. This step requires care and a jig made of amicroscope slide glued at 90 degrees on top of another microscope slideis a tool to help in the alignment. The slide also is be used to checkthe alignment on a microscope. The needle row (needle bundle) is tapedtogether with 50 micron thick tape. Two or more of the needle bundlesare stacked to form an electrode array with the tips of each bundlealigned with the next bundle. The needles are silver soldered to a wire,and alternating needle bundles are connected together electrically. Anoverall support structure is provided to support the electrode array ofneedle bundles.

[0162] As example of making an electrode assembly which includes longneedles is as follows. One embodiment of long needle electrodes can bemade in the following manner. Several ⅛ inch thick Lexan sheets are cutto the dimension of 1 cm by 1.5 cm. Two parallel rows are markded 5 mmapart. Drill points 2 mm apart are marked along each of the parallellines that are 5 mm apart. Holes are drilled at each marked point, 0.35mm in diameter.

[0163] Stainless-steel acupuncture needles, such as Seirin No. 8needles, are selected. Needles are inserted through one of the threeLexan pieces. Lengths of nickel capillary tube, 2 mm in length, are cutwherein the capillary tube has an inner diameter of 0.020 inches. A cutcapillary tube is slid onto each of the needles. Needles are insertedthrough the remaining two of the Lexan pieces. The Lexan piece nearestthe points is placed on a spacer that leaves 0.5 cm of needle extendingbeyond the Lexan piece. Using an epoxy adhesive, the needles are fixedin place to the Lexan piece nearest the point. The second piece is moved0.5 cm from the first piece. The space formed by three sides between thetwo Lexan pieces is sealed, and a high heat plastic or epoxy is pouredin to fill the space. The nickel pieces are soldered to the stainlesssteel by filling the space between the nickel tubing and the needle. Ahigh-voltage wire is soldered to connect one of the rows of needles atthe site of the nickel capillary tubing. A high-voltage wire is solderedto the other row of needles at the site of the nickel capillary tubing.The space between the last two Lexan pieces is sealed on three sides,and the space is filled with a high heat epoxy. The acupuncture needlehandles are removed, and the needles are rinsed to a point flush withthe top of the last Lexan piece. The top is sealed with an electricallyresistant epoxy. Banana plugs are placed on the other end of thehigh-voltage wires. The needles can be insulated by adding a smallamount of epoxy or Teflon paint to the ends of the needles. In addition,other selected areas of the needle may be insulated.

[0164] The long-needle electrodes can be coated in many ways. Thefollowing is one method. The needles are immersed in a solution of DNAthat has 0.3 micrograms per microliter of DNA in a TE buffer. Theneedles are suspended in the solution without touching the bottom of thecontainer. The container holding the liquid has a stainless-steel bottomthat serves as a negative electrode. All of the needles are thenconnected to a common positive pole of a power supply. A voltage of 1.5V is applied to the needles for several minutes. The needles are thenremoved from the solution and can be used immediately because the DNAforms a static coating on the electrodes. Alternatively the DNA can bedried on the needles. A statically-coated protectant such as sucrose maybe added to the needles by dipping in a sucrose solution prior to dryingthe DNA.

[0165] The capacity of the needles for retaining statically-coatedsurface DNA can be extended in several methods. One method is simply toapply multiple layers of statically-coated DNA to the surface of theneedles. Another method is to make the surface area of the needlegreater by adding a porous sintered metal surface. Another method is toadd a polymer or a gel to the electrode surface to provide athree-dimensional surface to the metal electrode surface for thestatically-coated DNA to adhere to.

[0166] The statically-coated DNA can be protected from the influence ofenvironmental DNAses by embedding the DNA in liposomes, adding DNAseinhibitors or coating the needle in a protectant. Sterile, desiccatedpackaging will also protect the statically-coated needles.

[0167] An example of using statically-coated, long needle electrodes isdescribed as follows. The needle electrodes containing thestatically-coated DNA are inserted into tissue for use. The smallvoltage electrophoretic potential may be applied for a period of secondsto minutes to move the DNA off of the needles and into the tissue.Electroporation pulses can then be added to drive the DNA into cells ofthe tissue. More electrophoretic pulses may be needed to help move theDNA into cells. After a resting period of several seconds, polaritiescan be reversed on the needles and the same protocol can be repeated.

[0168] In accordance with another aspect of the invention, polymers canbe used to modify adhesion of DNA to metal electrodes. As describedabove, DNA can be coated onto a metal electrode surface using electriccharge to facilitate the binding of the DNA to the electrode surface.However, such a DNA binding can be a high affinity binding which makesdriving the DNA off from the electrode using electric fields difficult.Consequently, in accordance with the invention, methods are provided forcontrolling the adhesion of DNA to fixed electrode surface 42 and easingthe release of DNA from the fixed electrode surface 42 using electricfields.

[0169] In accordance with an aspect of the invention, a water-insolubleadhesion controlling polymer 52 can be coated onto the electrodeunderbody 40 to form the fixed electrode surface 42. Then, the electrodereleasable molecules 44 are coated onto the fixed-surface adhesioncontrolling polymer 52. In such a case, when the electrodes are placednext to the biological cells, the water-insoluble adhesion controllingpolymer 52 remains fixed on the electrode underbody 40, and theelectrode releasable molecules 44, which are electric field separablemolecules, are driven off of the water-insoluble adhesion controllingpolymer 52.

[0170] Also, in accordance with an aspect of the invention, awater-insoluble adhesion controlling polymer 52 can be mixed togetherwith electrode releasable molecules 44, and the mixture can be coatedonto the electrode underbody 40 to form a fixed electrode surface 42which is intermingled with the electrode releasable molecules 44. Insuch a case, when the electrodes are placed next to the biologicalcells, the water-insoluble adhesion controlling polymer 52 remains fixedon the electrode underbody 40, and the intermingled electrode releasablemolecules 44, which are electric field separable molecules, are drivenoff of the water-insoluble adhesion controlling polymer 52 and driveninto the biological cells.

[0171] Alternatively, in accordance with the invention, the adhesioncontrolling polymer 52 can be water soluble and can be applied to thesurface of the electrode underbody 40 which serves as the fixedelectrode surface 42. Then, the electrode releasable molecules 44 arecoated onto the adhesion controlling polymer 52. In such a case, whenthe electrodes are placed next to the biological cells, thewater-soluble adhesion controlling polymer 52 is dissolved by the bodyfluids, and both the water-soluble adhesion controlling polymer 52 andthe electrode releasable molecules 44 are released from the fixedelectrode surface 42. Under the influence of applied electric fields,the electrode releasable molecules 44 are driven into the biologicalcells.

[0172] Alternatively, in accordance with the invention, the adhesioncontrolling polymer 52 can be water soluble and can be mixed with theelectrode releasable molecules 44. The mixture is applied to the surfaceof the electrode underbody 40 which serves as the fixed electrodesurface 42. In such a case, when the electrodes are placed next to thebiological cells, the water-soluble adhesion controlling polymer 52 isdissolved by the body fluids, and both the water-soluble adhesioncontrolling polymer 52 and the electrode releasable molecules 44 arereleased from the fixed electrode surface 42. Under the influence ofapplied electric fields, the electrode releasable molecules 44 aredriven into the biological cells.

[0173] Alternatively, all of the following can be used to coat theelectrode underbody 40: a water-insoluble adhesion controlling polymer52, a water soluble adhesion controlling polymer 52, and electrodereleasable molecules 44. In such a case, the water-insoluble moleculesremained fixed on the fixed electrode surface 42, and the water-solubleadhesion controlling polymer 52 and the electrode releasable molecules44 are released from the fixed electrode surface 42 when the electrodesare placed next to the biological cells. The electric fields then drivethe electrode releasable molecules 44 into the biological cells.

[0174] In general, in one class of methods, polymers are added to theelectrode underbody 40 prior to applying the electrode releasablemolecules 44, e. g. DNA. In another class of methods, polymers are mixedwith electrode releasable molecules 44, e. g. DNA, prior to coating theelectrode underbody 40 with the mixture.

[0175] For in vivo delivery of DNA, the polymer needs to bebiocompatible. A partial list of biocompatible polymers follows:poly(vinyl alcohol), poly(methacrylic acid), poly(ethoxazoline),poly(hydroxybutyrate), poly(caprolactone), poly(2-hydroxymethacrylate)and poly(acrylic acid). Co-polymers with components of these polymersmay also be biocompatible. Many other polymers are biocompatible and maybe appropriate for use.

[0176] Co-polymers may be especially useful if modified properties aredesired. For example, the addition of segments of cationic polymers innon-ionic polymers may promote binding of DNA to the polymer. Otherionic co-polymers may be used to impart an environmental sensitivity tothe polymer. Examples are pH sensitivity, temperature sensitivity,chemical sensitivity, solvent sensitivity or electric field sensitivity.All of these modifications can be used to control the release ofbiopolymers (DNA, RNA, proteins) mixed with or associated with theadditional polymer on the surface of an electrode.

[0177] One example of the use of a polymer to assist loading of DNA ontoa needle surface is as follows: Poly vinyl alcohol is dissolved in waterby heating. The amount of polymer used in this example is a 2% polymerin water (w/v). The electrode is dipped in the polymer and the electrodeis dried at 70 degrees centigrade. Next, 10 microliters of DNA at 2.5micrograms per microliter is added to the needle surface by pipetting.The DNA is dried onto the surface at 70 degrees centigrade. This processcan be done in one step by mixing the DNA and polymer prior toapplication. The needle electrode is then stored dry until used. Anotheradvantage of adding polymers to electrode metal surfaces with the DNA isto reduce electrolysis at electrode surfaces touching cells and DNA. Apolymer that remains on the metal surface as a hydrated polymer can keepthe metal separated from living cells and from the DNA. Such a polymerseparates electrode surfaces, which may have sites with active surfaceelectrolysis, from the living cells and from the DNA, thereby reducingthe chance of damage to either the cells or the DNA.

[0178] Even if polymers are used to assist in initial release ofbiopolymers, e. g. DNA, from the surface of the electrode, pulsedelectric fields are still needed for subsequent phases of the deliveryof the biopolymers into the biological cells. Pulses whose main purposeis electrophoresis may be used to move the biopolymer from next to theneedle electrode to surrounding tissue. An electroporation pulse isneeded to make the surrounding cells permeable to the biopolymers. Andan additional set of pulses, used for electrophoresis, may be needed tomove the biopolymer into the cells.

[0179] Although aspects of the invention have been described hereinabovein greater detail, here is a relatively brief review of aspects of theinvention.

[0180] A method is provided for delivery of molecules into biologicalcells. The method includes providing electrodes in an electrode assemblywherein the electrodes have fixed electrode surfaces which are coatedwith at least one static layer of electric field separable molecules tobe delivered. A waveform generator is provided for generating electricfields, and electrically conductive pathways are established between theelectrodes and the waveform generator. The electrode assembly having thestatically coated electrodes can be attached to an electrode assemblyholder for establishing electrically conductive pathways between theelectrodes and the waveform generator. The electrodes are located suchthat the biological cells are situated therebetween, and electric fieldsare provided in the form of pulse waveforms from the waveform generatorto the electrodes, such that molecules in the at least one static layerof the electric field separable molecules on the electrodes are drivenoff of the electrodes and delivered into the biological cells.

[0181] An electrode surface itself can serve as the fixed electrodesurface. The fixed electrode surface can include an oxidized metalsurface. The oxidized metal surface can include an oxide of silver, anoxide of nickel, or an oxide of copper. The fixed electrode surface caninclude fixed metal particles, or a roughened surface.

[0182] The electric field separable material on the fixed electrodesurface can include a gel coating, a solid layer of nonpolymericmaterial, or a polymer layer.

[0183] In accordance with another aspect of the invention, a method isprovided for immunotherapy which includes the steps of (a) obtainingstatically-coated electrodes which are statically-coated with animmuno-stimulating material, (b) inserting the statically-coatedelectrodes into a tissue to be treated, (c) applying electrophertic andelectroporation pulses to the statically-coated electrodes such that theimmuno-stimulating material is driven off of the statically-coatedelectrodes and into cells in the tissue.

[0184] In accordance with another aspect of the invention, an electrodeis provided which includes a coating having at least one static layer ofelectric field separable molecules to be delivered into biologicalcells. The electrode can include a fixed electrode surface which iscoated with the static layer of electric field separable molecules. Themolecules in the static coating can be in a solid phase, a gel.

[0185] The fixed electrode surface 42 can include a fixed surfacematrix, and the molecules in the static coating are in a liquid fixed onthe fixed surface matrix. The fixed surface matrix can include solidsurface particles. The solid surface particles can be metal particles.The fixed surface matrix can include a liposome matrix or a solidpolymer matrix.

[0186] The molecules in the static coating can be macromolecules, andthe macromolecules can include a polynucleotide vaccine, such as a solidphase polynucleotide vaccine, a DNA vaccine, a solid phase DNA vaccine,a RNA vaccine, or a solid phase RNA vaccine. The macromolecules in thestatic coating can include a protein-based vaccine, such as a solidphase protein-based vaccine. The macromolecules in the static coatingcan include an organ treating agent. The organ treating agent caninclude a deep tumor tissue treating agent.

[0187] The electrode can be in a form of a needle electrode.

[0188] In accordance with another aspect of the invention, the coatingof the electrode by the static coating molecules is carried out by thefollowing steps: preparing a liquid medium in which a quantity of themolecules are carried; contacting the electrodes with the preparedmedium; removing the electrodes from the medium; and drying off themedium, such that a static coating of the molecules remains on theelectrodes.

[0189] In accordance with another aspect of the invention, the coatingof the electrode by the static coating molecules is carried out by thefollowing steps: preparing a liquid medium in which a quantity of themolecules are carried; contacting the electrodes with the preparedmedium; applying pulse waveforms to the electrode, such that a portionof the molecules are bound to the electrode; removing the electrode fromthe medium; and drying off the medium, such that a coating of themolecules remains on the electrode.

[0190] In accordance with another aspect of the invention, an apparatusis provided for delivery of molecules into biological cells. Theapparatus includes a waveform generator which provides pulse waveforms.An electrode assembly holder is provided, and an electrode assemblyhaving a plurality of electrodes is mechanically supported by anelectrode assembly holder. The electrode assembly is electricallyconnected to the waveform generator through electrically conductivepathways. The electrode assembly includes electrodes which are coatedwith at least one static layer of molecules to be delivered into thebiological cells.

[0191] It is apparent from the above that the present inventionaccomplishes all of the objects set forth by providing a method and anapparatus for delivery of macromolecules into cells that do not causeskin damage that results in scarring. With the invention, a method andan apparatus for delivery of macromolecules into cells are providedwhich do not leave a residue of ballistic particles in cells that aretreated. With the invention, an electroporation method for deliveringmolecules to biological cells in the epidermis, near the basal lamina,does not have the treatment molecules pass through the skintransdermally. With the invention, a method and an apparatus fordelivery of macromolecules into cells are provided which do not employ ahypodermic needle. With the invention, a method and an apparatus fordelivery of macromolecules into cells are provided which do not employ afluid medium that flows down onto the electrodes as the electroporationprocess is being carried out on the patient.

[0192] With the invention, a method and an apparatus for delivery ofmacromolecules into cells are provided which do not employ a pressurizedfluid injection step for injecting fluid into a patient. With theinvention, relatively low absolute voltages are applied to cellsundergoing electroporation. With the invention, pulses that are appliedto the cells can have, if desired, relatively short pulse width to thecells undergoing electroporation. With the invention, a method and anapparatus for delivery of macromolecules into cells are provided whichcan employ, if desired, electrodes in which the base portions and tipportions of the electrodes are nonconductive. With the invention, amethod and an apparatus for delivery of macromolecules into cellsprovide disposable electrode assemblies. With the invention, a methodand an apparatus for delivery of macromolecules into cells are providedin which electrode assemblies are packaged in sterile packaging. Withthe invention, a method and an apparatus for delivery of macromoleculesinto cells are provided which permit treatment of tissues using coatedlong electrodes which have electric field separable material whichincludes a tissue treating agent.

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 25. An electrode whichincludes a coating having at least one static layer of electrodereleasable molecules to be delivered into biological cells:
 26. Aplurality of electrodes of claim 25 which form an electrode assembly.27. The electrode assembly of claim 26 wherein said plurality ofelectrodes are arranged in at least two parallel rows of electrodes. 28.The electrode assembly of claim 27 wherein said parallel rows ofelectrodes include needle electrodes.
 29. The electrode of claim 25wherein said molecules in said static coating are in a solid phase. 30.The electrode of claim 25 wherein said molecules in said static coatingare in a gel.
 31. The electrode of claim 25 wherein said electrodeincludes a fixed electrode surface which is coated with said staticlayer of electrode releasable molecules.
 32. The electrode of claim 31wherein: said fixed electrode surface 42 includes a fixed surfacematrix, and said molecules in said static coating are in a liquid fixedon said fixed surface matrix.
 33. The electrode of claim 31 wherein saidfixed electrode surface includes solid surface particles.
 34. Theelectrode of claim 33 wherein said solid surface particles are metalparticles.
 35. The electrode of claim 31 wherein said fixed electrodesurface includes a liposome matrix.
 36. The electrode of claim 31wherein said fixed electrode surface includes a solid polymer matrix.37. The electrode of claim 25 wherein said molecules in said staticcoating are macromolecules.
 38. The electrode of claim 25 wherein saidmacromolecules in said static coating include a polynucleotide vaccine.39. The electrode of claim 25 wherein said macromolecules in said staticcoating include a solid phase polynucleotide vaccine.
 40. The electrodeof claim 25 wherein said macromolecules in said static coating include aDNA vaccine.
 41. The electrode of claim 25 wherein said macromoleculesin said static coating include a solid phase DNA vaccine.
 42. Theelectrode of claim 25 wherein said macromolecules in said static coatinginclude an RNA vaccine.
 43. The electrode of claim 25 wherein saidmacromolecules in said static coating include a solid phase RNA vaccine.44. The electrode of claim 25 wherein said macromolecules in said staticcoating include a protein-based vaccine.
 45. The electrode of claim 25wherein said macromolecules in said static coating include a solid phaseprotein-based vaccine.
 46. The electrode of claim 25 wherein saidmacromolecules in said static coating include an organ treating agent.47. The electrode of claim 46 wherein said organ treating agent includesa deep tissue tumor treating agent.
 48. The electrode of claim 25 whichis in a form of a needle electrode.
 49. The electrode of claim 25,wherein coating of said electrode with said static layer of molecules tobe delivered to the biological cells is carried out by the followingsteps: preparing a liquid medium in which a quantity of said moleculesare carried, contacting said electrode with the prepared medium, andremoving said electrode from the medium and drying off the medium, suchthat a static coating of said molecules remains on said electrode. 50.The electrode of claim 25 wherein coating of said electrode with saidstatic layer of molecules to be delivered to the biological cells iscarried out by the following steps: preparing a liquid medium in which aquantity of said molecules are carried, contacting said electrode withthe prepared medium, applying pulse waveforms to said electrode, suchthat a portion of said molecules are bound to said electrode, andremoving said electrode from the medium and drying off the medium, suchthat a coating of said molecules remains on said electrode. 51.(Cancelled)